Dataset Information

Dynamic fluorescence depolarization: a powerful tool to explore protein folding on the ribosome.

ABSTRACT: Protein folding is a fundamental biological process of great significance for cell function and life-related processes. Surprisingly, very little is presently known about how proteins fold in vivo. The influence of the cellular environment is of paramount importance, as molecular chaperones, the ribosome, and the crowded medium affect both folding pathways and potentially even equilibrium structures. Studying protein folding in physiologically relevant environments, however, poses a number of technical challenges due to slow tumbling rates, low concentrations and potentially non-homogenous populations. Early work in this area relied on biological assays based on antibody recognition, proteolysis, and activity studies. More recently, it has been possible to directly observe the structure and dynamics of nascent polypeptides at high resolution by spectroscopic and microscopic techniques. The fluorescence depolarization decay of nascent polypeptides labeled with a small extrinsic fluorophore is a particularly powerful tool to gain insights into the dynamics of newly synthesized proteins. The fluorophore label senses both its own local mobility and the motions of the macromolecule to which it is attached. Fluorescence anisotropy decays can be measured both in the time and frequency domains. The latter mode of data collection is extremely convenient to capture the nanosecond motions in ribosome-bound nascent proteins, indicative of the development of independent structure and folding on the ribosome. In this review, we discuss the theory of fluorescence depolarization and its exciting applications to the study of the dynamics of nascent proteins in the cellular environment.

SUBMITTER: Weinreis SA

PROVIDER: S-EPMC2934862 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Dynamic fluorescence depolarization: a powerful tool to explore protein folding on the ribosome.

Weinreis Sarah A SA Ellis Jamie P JP Cavagnero Silvia S

Methods (San Diego, Calif.) 20100608 1

Protein folding is a fundamental biological process of great significance for cell function and life-related processes. Surprisingly, very little is presently known about how proteins fold in vivo. The influence of the cellular environment is of paramount importance, as molecular chaperones, the ribosome, and the crowded medium affect both folding pathways and potentially even equilibrium structures. Studying protein folding in physiologically relevant environments, however, poses a number of te ...[more]

PMID: 20685617

Similar Datasets

Project description:Although molecular simulation methods have yielded valuable insights into mechanistic aspects of protein refolding in vitro, they have up to now not been used to model the folding of proteins as they are actually synthesized by the ribosome. To address this issue, we report here simulation studies of three model proteins: chymotrypsin inhibitor 2 (CI2), barnase, and Semliki forest virus protein (SFVP), and directly compare their folding during ribosome-mediated synthesis with their refolding from random, denatured conformations. To calibrate the methodology, simulations are first compared with in vitro data on the folding stabilities of N-terminal fragments of CI2 and barnase; the simulations reproduce the fact that both the stability and thermal folding cooperativity increase as fragments increase in length. Coupled simulations of synthesis and folding for the same two proteins are then described, showing that both fold essentially post-translationally, with mechanisms effectively identical to those for refolding. In both cases, confinement of the nascent polypeptide chain within the ribosome tunnel does not appear to promote significant formation of native structure during synthesis; there are however clear indications that the formation of structure within the nascent chain is sensitive to location within the ribosome tunnel, being subject to both gain and loss as the chain lengthens. Interestingly, simulations in which CI2 is artificially stabilized show a pronounced tendency to become trapped within the tunnel in partially folded conformations: non-cooperative folding, therefore, appears in the simulations to exert a detrimental effect on the rate at which fully folded conformations are formed. Finally, simulations of the two-domain protease module of SFVP, which experimentally folds cotranslationally, indicate that for multi-domain proteins, ribosome-mediated folding may follow different pathways from those taken during refolding. Taken together, these studies provide a first step toward developing more realistic methods for simulating protein folding as it occurs in vivo.

Project description:BACKGROUND:Social networking sites, in particular Facebook, are not only predominant in students' social life but are to varying degrees interwoven with the medical curriculum. Particularly, Facebook groups have been identified for their potential in higher education. However, there is a paucity of data on user types, content, and dynamics of study-related Facebook groups. OBJECTIVE:The aim of this study was to identify the role of study-related Facebook group use, characterize medical students that use or avoid using Facebook groups (demographics, participation pattern, and motivation), and analyze student posting behavior, covered topics, dynamics, and limitations in Facebook groups with regards to educational usage. METHODS:Using a multi-method approach (interviews, focus groups, and qualitative and quantitative analysis of Facebook posts), we analyzed two representative Facebook groups of medical preclinical semesters at Ludwig-Maximilians-University (LMU) Munich. Facebook primary posts and replies over one semester were extracted and evaluated by using thematic content analysis. We developed and applied a coding scheme for studying the frequency and distribution of these posts. Additionally, we interviewed students with various degrees of involvement in the groups, as well as "new minorities," students not registered on Facebook. RESULTS:Facebook groups seem to have evolved as the main tool for medical students at LMU to complement the curriculum and to discuss study-related content. These Facebook groups are self-organizing and quickly adapt to organizational or subject-related challenges posed by the curriculum. A wide range of topics is covered, with a dominance of organization-related posts (58.35% [6916/11,853] of overall posts). By measuring reply rates and comments per category, we were able to identify learning tips and strategies, material sharing, and course content discussions as the most relevant categories. Rates of adequate replies in these categories ranged between 78% (11/14) and 100% (13/13), and the number of comments per post ranged from 8.4 to 13.7 compared with the average overall reply rate of 68.69% (1167/1699) and 3.9 comments per post. User typology revealed social media drivers (>30 posts per semester) as engines of group function, frequent users (11-30 posts), and a majority of average users acting rather as consumers or lurkers (1-10 posts). CONCLUSIONS:For the moment, the medical faculty has no active involvement in these groups and therefore no influence on accuracy of information, professionalism, and ethical issues. Nevertheless, faculty could in the future benefit by extracting relevant information, identifying common problems, and understanding semester-related dynamics.

Project description:CONSPECTUS: Several properties of nanomaterials, such as morphologies (e.g., shapes and surface structures) and distance dependent properties (e.g., plasmonic and quantum confinement effects), make nanomaterials uniquely qualified as potential choices for future applications from catalysis to biomedicine. To realize the full potential of these nanomaterials, it is important to demonstrate fine control of the morphology of individual nanoparticles, as well as precise spatial control of the position, orientation, and distances between multiple nanoparticles. In addition, dynamic control of nanomaterial assembly in response to multiple stimuli, with minimal or no error, and the reversibility of the assemblies are also required. In this Account, we summarize recent progress of using DNA as a powerful programmable tool to realize the above goals. First, inspired by the discovery of genetic codes in biology, we have discovered DNA sequence combinations to control different morphologies of nanoparticles during their growth process and have shown that these effects are synergistic or competitive, depending on the sequence combination. The DNA, which guides the growth of the nanomaterial, is stable and retains its biorecognition ability. Second, by taking advantage of different reactivities of phosphorothioate and phosphodiester backbone, we have placed phosphorothioate at selective positions on different DNA nanostructures including DNA tetrahedrons. Bifunctional linkers have been used to conjugate phosphorothioate on one end and bind nanoparticles or proteins on the other end. In doing so, precise control of distances between two or more nanoparticles or proteins with nanometer resolution can be achieved. Furthermore, by developing facile methods to functionalize two hemispheres of Janus nanoparticles with two different DNA sequences regioselectively, we have demonstrated directional control of nanomaterial assembly, where DNA strands with specific hybridization serve as orthogonal linkers. Third, by using functional DNA that includes DNAzyme, aptamer, and aptazyme, dynamic control of assemblies of gold nanoparticles, quantum dots, carbon nanotubes, and iron oxide nanoparticles in response to one or more stimuli cooperatively have been achieved, resulting in colorimetric, fluorescent, electrochemical, and magnetic resonance signals for a wide range of targets, such as metal ions, small molecules, proteins, and intact cells. Fourth, by mimicking biology, we have employed DNAzymes as proofreading units to remove errors in nanoparticle assembly and further used DNAzyme cascade reactions to modify or repair DNA sequences involved in the assembly. Finally, by taking advantage of different affinities of biotin and desthiobiotin toward streptavidin, we have demonstrated reversible assembly of proteins on DNA origami.

Dataset Information

Dynamic fluorescence depolarization: a powerful tool to explore protein folding on the ribosome.

Publications

Dynamic fluorescence depolarization: a powerful tool to explore protein folding on the ribosome.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets